Akihiko Nii

Production of IL-1 and its receptor antagonist is regulated differently by IFN-gamma and IL-4 in human monocytes and alveolar macrophages

Interleukin-4 (IL-4) has previously been found to downregulate interleukin-1 (IL-1) production, but to upregulate the production of IL-1 receptor antagonist (IL-1ra) in human monocytes stimulated with lipopolysaccharide (LPS). In the present study we wanted to determine whether the production of IL-1ra in human monocytes and alveolar macrophages (AMs) is regulated differently at the protein and messenger ribonucleic acid (mRNA) levels by IL-4 and interferon-gamma (IFN-gamma). AMs and monocytes obtained from healthy donors by bronchoalveolar lavage and centrifugal elutriation were stimulated with LPS in the presence or absence of IL-4 or IFN-gamma, and the expression of mRNA for IL-1 and IL-1ra was measured by Northern blot analysis. The production of IL-1 and IL-1ra was quantitated by enzyme immunoassays (EIAs). Spontaneous IL-1ra production was seen in AMs after incubation for 4 h in medium alone, but not in blood monocytes, at both the protein and mRNA levels. The spontaneous expression of the IL-1ra gene in AMs was augmented by incubation with IL-4. Interleukin-1 beta (IL-1 beta) production by LPS-stimulated AMs and monocytes was upregulated by IFN-gamma, but downregulated by IL-4. Interestingly, when stimulated with LPS, IFN-gamma inhibited IL-1ra production by monocytes, but up-regulated its production in human AMs at the protein and mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Autonomous Expressions of Cytokine Genes by Human Lung Cancer Cells and Their Paracrine Regulation

Cell‐to‐cell interaction between tumors and host inflammatory cells is important for the subsequent cancer progression or regression. We examined the expressions of mRNAs for various proinflammatory cytokines by nine human lung cancer cell lines and the influences of cytokines on their gene expressions. The cytokines used were interleukin 1β (IL‐1β), interleukin 6 (IL‐6), tumor necrosis factor α (TNF‐α), granulocyte‐macrophage colony stimulating factor (GM‐CSF) and monocyte chemotactic and activating factor. Gene expressions of cytokines were measured by Northern blot analysis. Substantial expressions of cytokine genes were detected in several lung cancer cell lines such as RERF‐LC‐MS, RERF‐LC‐OK and VMRC‐LCD, although the levels of expression of each cytokine varied in different cell lines. Four lung cancer cell lines (RERF‐LC‐MS, RERF‐LC‐OK, A549 and YO‐88) were used to examine the effects of exogenous cytokines (IL‐1β, TNF‐α and GM‐CSF) on cytokine gene expressions by the cells. TNF‐α and IL‐1β caused significant changes in the levels of mRNA expressions of certain cytokines. Moreover, on stimulation with TNF‐α, RERF‐LC‐OK cells produced IL‐6 extracellularly. These extensive differences in the levels of gene expressions and productions of cytokines could have profound effects on the interactions between human lung cancer cells and the corresponding host cells.

Induction of a 26-kDa membrane-form tumor necrosis factor (TNF)-α in human alveolar macrophages

The expressions of a membrane form of TNF (m‐TNF) by human alveolar macrophages (AM) and autologous blood monocytes from healthy donors were examined. Upon lipopolysaccharide (LPS) stimulation, AM produced 26‐kDa TNF‐α on their cell surface. We designed a bioassay for measuring m‐TNF in which macrophages were fixed with paraformaldehyde after stimulation for 18 h, then m‐TNF activity was assessed as cytotoxicity of fixed macrophages on L929 cells. This assay was specific to m‐TNF because: 1) no soluble factors were contributed to the cytotoxicity of fixed AM, 2) anti‐TNF‐α monoclonal antibody completely neutralized m‐TNF activity, and 3) m‐TNF activity was not altered after low‐ pH or high‐salt treatment. On LPS stimulation, AM produced significant amounts of m‐TNF earlier than TNF‐α secretion. AM also expressed significant amounts of m‐TNF when stimulated with other bacterial components and their derivatives. Interleukin (IL)‐4 suppressed both m‐TNF production and TNF‐α secretion. p‐Toluene‐ sulfonyl‐L‐arginine methyl ester (TAME) inhibited specifically TNF‐α secretion and not m‐TNF expression. Although blood monocytes produced small amounts of m‐ TNF, monocyte‐derived macrophages showed enhanced m‐TNF after cultivation with GM‐CSF for 10 days. These findings indicate that m‐TNF is expressed as a step in the TNF‐α producing system, and suggest that m‐TNF may play important roles in exhibition of macrophage function in situ.

Lymphokine-activated Killer Induction and Its Regulation by Macrophages in Malignant Pleural Effusions

Mononuclear cells (MNC) from pleural effusions and peripheral blood of 18 patients with primary lung cancer with malignant pleural effusion were studied. Pleural and blood MNC generated lymphokine‐activated killer (LAK) activity similarly when cultured for 4 days with an optimal concentration of interleukin 2 (IL‐2). Highly purified lymphocytes (>98%) and monocyte‐macrophages (>90%) were isolated by discontinuous Percoll gradient centrifugation from pleural and blood MNC. Pleural macrophages, as well as blood monocytes, showed significant augmenting effects on in vitro LAK cell induction from pleural and blood lymphocytes by IL‐2. During daily intrapleural administration of IL‐2, significant induction of LAK activity in vivo was observed after 3 days, but then this LAK activity in pleural MNC decreased almost to zero by day 15. Daily injections of IL‐2 resulted in reduction in the up‐regulation of LAK induction by pleural macrophages and also in increase in the levels of soluble IL‐2 receptors in pleural effusions. These findings indicate that in vivo LAK induction of lymphocytes in malignant effusions by IL‐2 may be regulated by macrophages in the effusions.

Differential effects of interleukin‐4 on superoxide anion production by human alveolar macrophages stimulated with lipopolysaccharide and interferon‐γ

The effect of interleukin‐4 (IL‐4) on the activation state of human alveolar macrophages (AMs) and blood monocytes induced by lipopolysaccharide (LPS) or recombinant interferon‐γ (IFN‐γ) was investigated on the basis of their ability to produce superoxide anion (O2 ‐). AMs were obtained from healthy donors by bronchoalveolar lavage, and O2 ‐ productions of these cells were assayed by a cytochrome c reduction method after incubation with stimulants for 24 h. AMs produced more O2 ‐than autologous blood monocytes when stimulated with LPS. IL‐4 alone had little effect on O2 ‐ production by unstimulated AMs but down‐regulated O2 ‐ production by LPS‐stimulated AMs in a dose‐dependent manner. IL‐4 also suppressed O2 ‐ production by AMs induced by the synergistic actions of muramyl dipeptide (norMDP) and IFN‐γ. Maximum suppression by IL‐4 of O2 ‐ production by AMs was observed when IL‐4 was added within 1 h after initiation of LPS stimulation. AMs also showed high O2 ‐ production when stimulated with IFN‐γ alone. In contrast to its suppression of O2 ‐ production by LPS‐stimulated AMs, IL‐4 enhanced O2 ‐ production by AMs stimulated with IFN‐γ. These data suggest that IL‐4 is an important regulator of O2 ‐ production by macrophages through different pathways depending on the stimulus.

IL‐6 in malignant pleural effusions and its augmentation by intrapleural instillation of IL‐2

The levels and activities of endogenous IL‐6 in malignant pleural effusions due to lung cancer before and during daily intrapleural instillations of recombinant IL‐2 were examined by enzyme immunoassay and bioassay using an IL‐6‐dependent murine hybridoma cell line MH60.BSF2. Before therapy, malignant pleural effusions contained various levels of IL‐6. Daily intrapleural instillation of recombinant IL‐2 for treatment of malignant pleurisy resulted in significant augmentation of the levels and activities of IL‐6 in the pleural effusions. On fractionation of the pleural effusion by chromatography. one major peak of material with a mol. wt of 24 kD showed IL‐6 activity. In contrast, no significant level of tumour necrosis factor‐alpha or IL‐1β was detectable in pleural effusions before or during local IL‐2 therapy. These data suggest that IL‐2 is an important regulatory factor of secondary IL‐6 production.

Add-on effects of suplatast tosilate in bronchial asthma patients treated with inhaled corticosteroids

Th2 cytokines play an important role in the pathogenesis of asthma. In the present study, we investigated the effect of suplatast tosilate, a selective Th2 cytokine inhibitor, on asthma control, in terms of subjective symptoms and pulmonary function in patients treated with inhaled corticosteroids. Thirty-eight patients with bronchial asthma being treated with inhaled corticosteroids were given suplatast tosilate (100 mg three times daily) for 12 weeks, in a multicenter setting. During the study period, other medications were continued. Morning and evening peak expiratory flow, asthma symptoms, blood eosinophil count and serum IgE levels were monitored. Suplatast tosilate treatment was associated with a significant improvement in mean morning peak expiratory flow (from 295 L/min to 348 L/min, P < 0.01) and evening peak expiratory flow (from 313 L/min to 357 L/min, P < 0.01). The mean daily variation in peak expiratory flow was significantly reduced (from 11.6% to 7.3%, P < 0.01) by suplatast tosilate treatment. The greatest improvement in peak expiratory flow was observed in patients whose blood eosinophil counts were decreased by suplatast tosilate treatment. Treatment with suplatast tosilate improved pulmonary function in patients with bronchial asthma. Our results suggest the therapeutic effects observed may occur through suppression of eosinophilic inflammation.

Augmentation by transferrin of IL-2-inducible killer activity and perforin production of human CD8+ T cells.

The effects of human transferrin (Tf) on lymphokine (IL‐2)‐activated killer (LAK) induction from blood lymphocytes of healthy donors was examined. LAK cells were induced by 6‐day incubation in medium with recombinant human IL‐2 of lymphocytes, and their cytotoxic activity was assessed by measuring 51Cr release from NK‐resistant Daudi cells. Tf alone did not induce any LAK activity, but in combination with IL‐2, it augmented LAK induction dose‐ and time‐dependently. This augmenting effect was completely abolished by pretreatmenl with anti‐Tf antiserum. Tf augmented the proliferative response of lymphocytes to IL‐2 and their expressions of receptors for IL‐2 and Tf. CD8+ T cells were isolated from purified blood lymphocytes using antibody‐bound magnetic beads. Addition of Tf to cultures of CDS+ cells resulted in significant augmentation of killer cell induction and perforin (PFP) production after 4 days stimulation with IL‐2. These results indicate that Tf is important in generation of IL‐2‐inducible killer properties and PFP activity of human CD8+ T cells.

Enhancement by Monocytes of Perform Production and Its Gene Expression by Human CD8+ T Cells Stimulated with Interleukin‐2

Pore‐forming protein (PFP) is an important effector molecule for cytotoxicity mediated by cytotoxic T cells and NK cells. In the present study, the effect of monocytes on PFP production by inter‐leukin‐2 (IL‐2)‐stimulated T lymphocytes was examined. Highly purified lymphocytes (>99%) and monocytes (> 90%) were isolated by centrifugal elutriation from peripheral blood of healthy donors, and, CD4+ and CD8+ cells were isolated from the purified lymphocytes by using antibody‐bound magnetic beads. PFP production was quantitated with a universal microspectrophotometer in combination with immunostaining using anti‐PFP antibody. Monocytes did not produce any PFP. High levels of PFP production were observed in CD8+ cells, but not CD4+ cells after incubation for 4 days with IL‐2. Addition of monocytes to cultures of CD8 + cells resulted in significant augmentation of PFP production after 3 days’ stimulation with IL‐2. Monokines (TNFα and IL‐6) caused a significant increase in PFP production by IL‐2‐stimulated CD8+ cells. Northern blot analysis revealed that the PFP mRNA level was enhanced by stimulation with IL‐2, and that addition of monocytes to cultures of CD8 + cells plus IL‐2 augmented their PFP mRNA expression. These observations suggest that monocytes are important in in situ regulation of the CD8 + T cell‐mediated cytotoxic response through production of PFP.

Synergism of synthetic acyltripeptide and its analogs with recombinant interferon γ for activation of antitumor properties of human blood monocytes

SummaryHuman blood monocytes freshly isolated by centrifugal elutriation from healthy volunteers were not cytotoxic to allogeneic A375 melanoma cells, but they were activated to the tumoricidal state by incubation in vitro with FK-565, (heptanoyl-γ-D-Glu-(L)-meso-α,ε-A2pm(L)-D-AlaOH), which is a synthetic acyltripeptide closely resembling cell wall peptidoglycan peptides of Streptomyces in structure. Among 11 different derivatives of FK-565, 7 analogs were more potent activators of monocytes for tumor cell killing than FK-565. The maximal expression of tumoricidal nonocytes was dependent on the concentration of FK-565 or its analogs added and the ratio of monocytes to target tumor cells. In a parallel experiment, a combination of a subthreshold concentration of FK-565 or its analogs (FR-42148 and FR-42149) and recombinant interferon γ (rIFN-γ) induced significant monocyte-mediated tumorcell killing, indicating that the effects of rIFN-γ and acyltripeptide or its analogs in monocyte activation are synergistic. In contrast to rIFN-γ, recombinant rIFN-αA and rIFN-β had additive effects with acyltripeptide or its analogs in human monocyte activation. These results suggested that synthetic acyltripeptide and its analogs combined with rIFN-γ could be of clinical value for in situ activation of the tumoricidal activity of human blood monocytes responsible for eradication of cancer metastases.